The present invention relates to the field of selective inhibition of androgen receptor. The invention further relates generally to the field of gene therapy, and particularly gene therapy in the treatment of prostatic cancer.
The prostate gland is an androgen-dependent organ and continues to grow with age. This leads to enlarged prostate in older men with consequent pathological manifestations. Androgen receptor is the principal mediator of prostatic growth.
A hammerhead ribozyme is a small RNA capable of cleaving a target RNA in a catalytic manner in the presence of a divalent cation (Pyle, 1993). Naturally occurring hammerhead ribozymes were discovered in certain plant viroids and viruses (Forster and Symons, 1987). The hammerhead ribozyme acts in xe2x80x9ccisxe2x80x9d during viral replication by the rolling circle mechanism. However a hammerhead ribozyme was engineered to cleave in xe2x80x9ctransxe2x80x9d against other RNAs (Uhlenbeck, 1987). A hammerhead ribozyme consists of antisense segments (stems I and III) and a catalytic domain (stem II). It can be designed to target specific mRNAs by selecting sequences flanking the catalytic element. The only requirement for the target substrate is the sequence HUX (H can be any nucleotide, X is A, C or U), where cleavage occurs after X (Haseloff and Gerlach, 1988). Hammerhead cleavage produces RNA products with 5xe2x80x2 hydroxyl and 2xe2x80x2, 3xe2x80x2 cyclic phosphate termini (Buzayan, et al., 1986; Prody, et al., 1986). A hammerhead ribozyme has potential therapeutic applications, e.g., it inactivates specific RNAs in vivo, such as HIV-1 gene expression (Sarver, et al., 1990; Ojwang, et al, 1992; Yu, et al., 1993), RNAs responsible for other viral infections (Chen, et al., 1992; Sullenger and Cech, 1993; Tang, et al., 1994) and the RNA transcripts of other genes (Scanlon, et al., 1991; Kashani-Sahetet, et al., 1992; Lange, et al., 1993; Ha and Kim, 1994; Kobayashi, et al., 1994; Sioud, et al., 1994; Jarvis, et al., 1996; Ohta, et al., 1996; Sioud, 1996).
Androgen receptor (AR) is a ligand-activated transcription factor belonging to the steroid/thyroid hormone receptor superfamily (Evans, 1988; Beato, 1989). AR plays an important role in the coordination of the male-specific sexual phenotype and in the development of the male-reproductive organs such as the prostate gland (Quiley, et al., 1995). AR is expressed in various cells and tissues (Chang, et al., 1995; Roy and Chatterjee, 1995). It has also been considered as an etiologic factor for human benign prostatic hyperplasia (Brolin, et al., 1992; Wilding, 1992; Lepor, et al., 1993). Furthermore, AR gene mutations are involved in primary and secondary prostate cancer (Newmark, et al., 1992; Culig, et al., 1993; Suzuki, et al., 1993; Taplin, et al., 1995). A high expression of AR in recurrent prostate cancer cells and metastatic prostate cancer cells has also been observed (Taplin, et al., Viskarpi, et al., 1995; Umeki, et al., 1996). However, how the AR regulates differentiation and development of the male reproductive organs and its role in prostatic diseases are not known.
Clinical treatment of prostatic cancer has included the use of surgical techniques to remove enlarged prostate tissue, or the use of enzyme inhibitors such as PROSCAR(trademark). PROSCAR inhibits 5-alpha reductase, which is the enzyme that converts testosterone to dihydrotestosterone. The abolition of testesterone itslf to induce androgen action limits the use and effectiveness of this therapy. These approaches are thus undesirable in many patients. Need continues to exist in the medical arts for a therapy that provides a more targeted approach to treatment of this pathology.
Androgen receptor plays a central role in the development, differentiation and maintenance of the male reproductive organs (Coffey, 1988; Griffin et al., 1989; Migeon et al., 1994). It is also involved in prostate disorders and other diseases (Edward, 1992; Macke et al., 1993; Qingley et al., 1995). The molecular mechanisms whereby AR regulates the physiological and pathological events are not clearly understood (Wilding, 1992; Lapor and Lawson, 1993). Hence, there has been no significant development of clinical approaches for treatment of prostate and related disorders.
The present invention describes inactivation of AR gene expression by engineering hammerhead ribozymes to cleave specific sites in AR mRNA. The present in vitro studies of hammerhead ribozymes reveal a high efficiency of such cleavage activity. The hammerhead ribozymes suppress AR mRNA expression in cultured cells.
Included in the present invention are hammerhead ribozymes that can selectively and efficiently degrade human androgen receptor messenger RNA. Also part of the present invention are expression vectors containing the gene for a ribozyme that, when introduced into a human prostate cancer cell, is capable of abolishing the androgen receptor mediated transactivation of a reporter gene. Targeting of the ribozyme gene into specific tissues of transgenic mice can be done to produce tissue-specific inactivation of androgen receptor. Therapeutic use of the ribozymes of the present invention to suppress androgen action in human clinical conditions such as the prostatic hyperplasia may be accomplished in vivo of the present invention.
The following provides three selection criteria in identifying and designing synthetic ribozyme of the present invention: 
Based on these three selection criteria, the inventors designed three hammerhead ribozymes and tested their effectiveness in the in vitro endonuclease assay. One of these ribozymes, HR-2 was found to be particularly highly effective in selectively degrading the androgen receptor mRNA. This androgen receptor degrading ribozyme is more active than all ribozymes reported in the literature. 
After eliminating sequence regions that can potentially form secondary structures or have significant homology to heterologous mRNAs, the inventors chose three structural domains of the AR mRNA with high AU contents as targets for the hammerhead ribozyme.
Hammerhead ribozymes are composed of two functionally distinct components; (i) the central catalytic core usually containing about 24 nucleotides with a conserved stem loop structure, and (ii) two variable specifier sequences on both 5xe2x80x2 and 3xe2x80x2 sides of the catalytic core that are complementary to the target RNA. The three targeted areas on the AR mRNA that were selected correspond to (i) transactivation domain of the rat AR (ribozyme, R-1), (ii) transactivation domain of the human AR (ribozyme, H-1), and (iii) the DNA binding domain of the human AR with 95% homology to the rat AR (ribozyme, HR-2). All three of these ribozymes contained 9-12 nt long specifier arms on each side of the catalytic core. Both ribozymes and truncated AR targets were cloned into the Bluescript vector and were transcribed with either T7 or T3 RNA polymerase for the in vitro endonuclease assay. At an equimolar enzyme-substrate ratio and at 37xc2x0 C., R1 and H1 required xcx9c4 hr for 75 to 100% cleavage of the substrate. The ribozyme HR-2 required less than 30 min for complete cleavage of the target substrate. The HR-2 ribozyme was also effective at a E:S ratio as low as 1:50. A mutant HR-2 containing two base substitutions within the catalytic core was enzymatically inactive and the wild type HR-2 did not act on substrates corresponding to R-1 and H-1, substantiating the specificity of the ribozyme function.
The inventors examined the effectiveness of the HR-2 in AR(xe2x88x92) PC3 (prostate cancer derived) cells transfected with the AR expression vector and a reporter construct containing MMTV-CAT. In this transfection assay an expression vector containing the HR-2 ribozyme was able to inhibit the AR mediated transactivation of the MMRV-promoter in a dose-dependent manner with a more than 95% inhibition at an AR:HR-2 ratio of 100. These results indicate that ribozymes can be an effective means for inactivating androgen action and are useful as a therapeutic agent when delivered to the target tissue through expression vectors and tissue-specific promoters.
By means of selection of the target base compositions (A-T, G-C pairs), the optimum size of the two sided arms, and the in vitro testing of various ribozyme constructs, the inventors have produced particular synthetic ribozymes having high activity and specificity for the human androgen receptor mRNA.
The inventors have developed the specifically active ribozyme HR-2 (SEQ ID NO:2) that cleaves the human androgen receptor mRNA (SEQ ID NO:1) at base positions 2374/2375 (Table 1). The nucleotide structure of the rat ribozyme (SEQ ID NO:1) and its complementarity to the rat androgen receptor mRNA target site (SEQ ID NO:10) are as follows: 
The in vitro cleavage of AR mRNA sequence by the ribozyme and kinetics of the endonuclease activity have established utility of the present invention.
Mammalian expression vectors containing the ribozyme HR-2 and a RNA polymerase II promoter derived from the cytomegalovirus (CMV) or a RNA polymerase III promoter derived from the gene for a small nuclear RNA (U6 RNA) when cotransfected into human (PC-3, prostate cancer derived) and rodent (3T3, mouse fibroblast derived) cells showed a dose-dependent inactivation of androgen receptor function.
This involved establishing:
i) Cell transfection system.
ii) Inhibition in the human prostate cancer cells by the CMV construct; and
iii) Inhibition in the NIH 3T3 cells by the U-6 construct
A transgenic mouse line containing selective over-expression of the androgen receptor in the liver targeted by the liver-specific phenylalanine hydroxylase gene promoter has been created by the present inventors. The same promoter is being used to target the HR-2 ribozyme to the liver. The homozygous AR transgenic mouse will be crossed with the HR-2 transgenic mice and the hepatic level of the androgen receptor in the hybrid mice will be monitored.
Gene therapy to suppress prostatic hyperplasia during old age and to destroy aberrant forms of androgen receptor mRNA in prostate cancer cells is thus available. This may involve therapeutic use of the ribozyme to suppress prostatic hyperplasia. Such can be performed by local delivery of the ribozyme gene construct inserted into any one of the emerging in vivo gene delivery vectors (for the most recent development see, Naldini et al., 1996) during cauterization of the enlarged prostate. At an advanced stage of prostate cancer the androgen receptor undergoes mutation and begins to function independent of the androgenic ligand (Taplin et al., 1995). Presently no specific therapeutic means to inhibit such an androgen-independent form of the receptor is available. The HR-2 ribozyme inserted into the appropriate delivery vector can be an effective drug to control such androgen independent mutant form of the receptor and to inhibit the resultant neoplastic prostate cell growth.
The present inventors demonstrate that two hammerhead ribozymes are able to cleave the RNA immediately following the GUC triplet sequences at positions 1393 and 2209 of the AR mRNA, respectively. Compared to a variety of other triplets, the GUC triplet preceding a particular site on the RNA substrate makes that site much more efficiently cleaved. (Haseloff and Gerlach, 1988; Ruffner et al., 1990; Shimayama et al., 1995; Hendrix et al., 1996). It can be demonstrated that the RNA phosphodiester bond immediately following the nucleotide residue, which has its ribose sugar group held in a south conformation (that is C2.,-endo xe2x80x94C3,-exo) is most preferably cleaved by the hammerhead ribozyme (Plavec et al., 1994). Furthermore, compared to other nucleotidyl 3xe2x80x2-ethylphosphates, cytidine 3xe2x80x2-ethylphosphate can most readily assume the south conformation at the ribose moiety, thus explaining the preference for C at the third base of the triplet preceding the cleavage site (Plavec at el., 1994). That G is the preferred first base in the marker triplet follows from the analysis of Kcat and Km of the cleavage reactions using substrate in which the first base is changed from G to another base (Shimayama et al, 1995). The base preference at the first position of the triplet, despite its distance from the cleavage site, indicates that the entire triplet contributes to the structure of the transition state intermediate formed during the phosphodiester bond cleavage reaction (Hendrix et al, 1996).
Specificity and efficiency are also important parameters to consider in designing a hammerhead ribozyme. Factors, such as the secondary structure of the substrate and length as well as composition of the flanking sequences (stems I and III) of hammerhead ribozymes affect function. Many studies have shown that a hammerhead ribozyme targeted to a predicted open stemloop structure within target RNA substrate is more effective in catalyzing cleavage of the RNA substrate when it targets a base paired region (L""Huillier et al., 1992; Steinecke et al., 1994; Hendrix et al., 1996; Lieber and Strauss, 1996). Christoffersen and Marr found that this criterion applies well to ribozyme activity in cell culture and animal studies (Christoffersen and Marr, 1995). The length and composition of flanking sequences of a hammerhead ribozyme are also important factors in optimizing a designed hammerhead ribozyme. Although the length of the flanking sequences of the hammerhead ribozyme varies in different target sites, optimal cellular efficiency is observed with relatively short sequences of between 10-20 residues (Fedor et al., 1990; Herschlog, 1991; Heidenreich and Eckstein, 1992; Ferbeyre et al., 1996; Jarvis et al., 1996). Up to a point (xcx9c25 residues), longer flanking sequences can increase specificity of the hammerhead ribozyme, but it also decreases the cleavage efficiency, due to a decrease in turnover of the ribozyme (Heidenreich and Eckstein, 1992; Bertrand et al., 1994; Ferbeyre et al., 1996). This is supported by the further finding that reduced length of the flanking sequences between substrate and hammerhead ribozyme increases the rate of cleavage (Goodchild and Kohli, 1994). Composition of the flanking sequences is another consideration. A target region of RNA substrate with a high number of G or C residues so stabilizes interaction between the target and ribozyme that their separation after cleavage may be deterred (Bertrand et al., 1994). It is therefore preferable to select A/U rich flanking sequences since A:U base pair is weaker than G:C. Additionally, A-rich sequences in the flanks of the hammerhead ribozyme avoid the possibility of U-G wobble base pairing that can decrease discrimination between target sites (Hersalag, 1991; Bertrand et al., 1994). The MFOLD program was used to study the secondary structure of AR mRNA (Zuker and Stiegler, 1981; Zuker, 1989). Two cleavage sites of AR mRNA with open-loop or single-stranded regions were identified. The open regions contain GUC triplet sequences flanked by U-rich sequences that are not homologous to other gene sequences. Flanking sequences with 19 nucleotides and 18 nucleotides (stems I and III) were selected for HI and HR2 hammerhead ribozymes (FIG. 1) that contain 58% and 61% A/U-rich sequences, respectively. In the assay system, these hammerhead ribozymes were highly specific and catalyzed cleavage of only the AR mRNA substrate (FIG. 7).
Highly specific hammerhead ribozyme activity has been observed in cultured cells, and in animals (Saxena and Ackerman, 1990; Sullenger and Cech, 1993; Yu et al., 1993; Larsson et al., 1994; L""Hulillier et al., 1996). L""Hulillier et al. (1996) have observed that a hammerhead ribozyme cleaves only exogenous xcex1-lac mRNA against which it was designed, and not against endogenous xcex1-lac mRNA in transgenic mice, indicating the specificity of designed hammerhead ribozyme. In addition to these demonstrated specificities, the cleavage rate of both the HI and HR2 ribozymes is rapid and complete within 30 min at 1:1 molar ratio of ribozyme: substrate. However, compared to HI, HR2 is more efficient in vitro (FIG. 5) and in vivo (FIG. 8). The reason for the higher activity of HR2 over HI is not clear. One explanation could be that the target region in the AR mRNA for HR2 is more exposed than the target region for HI, so that HR2 has a better access to its RNA substrate (Kobayashi et al., 1994).
The following table enumerates several sequences that were used in the testing or development of the present invention.